Project Summary Despite recent advances, melanoma remains the deadliest form of skin cancer, and new therapeutic strategies are urgently needed. The MAP kinase (MAPK) and PI3K/AKT pathways are central to both disease progression and chemoresistance in melanoma, with dual targeting approaches yielding promising results that would benefit from increased target specificity and selectivity. The AKT family of serine/threonine kinases comprises three highly homologous and functionally distinct isoforms (AKT1, AKT2, and AKT3) that play unique roles in melanoma but have not yet been leveraged for isoform-specific targeting. AKT2 is an attractive candidate for therapeutic intervention, given that AKT2 mutations and AKT2 gene amplification are known mechanisms of adaptive resistance to current targeted therapies such as the BRAF inhibitor (BRAFi) Vemurafenib. We hypothesize that AKT2 is involved in melanoma invasion and metastasis, and acts as a mediator of the BRAFi response. We further suggest that testing AKT2 isoform-specific targeting in combination with BRAF inhibitors is an anti-melanoma strategy that may avoid serious dose limiting toxicities commonly associated with pan-AKT inhibition. This work will establish the contribution of AKT2 to BRAF-mutant melanoma progression and metastasis, using both human melanoma cell lines and mouse models. In Aim1, we will investigate the effect of AKT2 knockdown on human melanoma cell migration and invasion, and whether this differs in the presence of the BRAFi Vemurafenib. Further, we will identify AKT2-specific substrates that influence the BRAFi response by immunoprecipitating phosphoproteins reacting with AKT phospho-substrate-specific antibodies in cells treated with Vemurafenib with or without AKT2-specific knockdown. Immunoprecipitates will be analyzed by mass spectrometry, prioritized and validated using AKT2 wild-type and knockdown cells to determine if manipulation of any substrate alters cell sensitivity to BRAF inhibition. In Aim 2, we will investigate the contribution of genetic AKT2 loss in a BRAF-mutant melanoma prone mouse model, and perform metastasis seeding experiments by intra-cardiac injection of AKT2 null cell lines, as well as drug studies in allograft tumors. We will investigate the effect of co-targeting BRAF and AKT2 with specific inhibitors, as a proof-of- principle for future studies. With AKT inhibitors in clinical trials for melanoma, our findings could yield novel therapeutic strategies and increase the efficiency of existing therapies to improve treatment options and outcome for this devastating disease.